Method of screeing for pathogens

ABSTRACT

Disclosed is a method of screening for the presence of MRSA in a subject, the method comprising the steps of:
         (a) obtaining a suitable sample from the subject using a swab or other suitable sample collection device;   (b) substantially immediately after performing step (a), contacting the swab or other sample collection device with an identification medium, said medium comprising:
           (i) nutrients to allow the growth of MRSA;   (ii) at least one inhibitor substance to inhibit preferentially the growth of organisms other than MRSA; and   (iii) a visual indicator substance which generates a visual indication of the growth of MRSA if present;   
           (c) incubating the identification medium, typically whilst still in contact with the swab or other sample collection device, in conditions which are sufficient to cause the growth of MRSA; and, after sufficient time has elapsed;   (d) inspecting the medium to determine the presence or absence of the visual indication of the growth of MRSA.

CROSS-REFERENCES TO RELATED APPLICATION

This application claims priority based on German Patent Application No.GB 0814099.8, filed Aug. 1, 2008, of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to a method of screening for pathogens, inparticular to a rapid method of screening for the presence ofmeticillin-resistant Staphylococcus aureus (MRSA), and to media and kitsof use in such methods.

BACKGROUND OF THE INVENTION

Nosocomial or iatrogenic infection by MRSA is a well-known andwidespread problem in the UK and elsewhere. In an effort to help controlthe incidence of MRSA infections in the UK, it is planned to screenevery patient entering hospital, in order to identify those individualswho may be asymptomatic carriers. It is estimated that between 10 and20% of the general population in the UK may be carriers of MRSA.

Currently, conventional methods of screening entail taking a swab (suchas a nasal swab) from an individual at the point of care. The swab isthen placed in a sterile container and forwarded to the microbiology labin the hospital, where it is unpackaged and used to inoculate ascreening plate. The plate contains a solid medium which has ingredientsto stimulate the growth of any MRSA bacteria present, but also containssome sort of marker which gives a visible indication if MRSA arepresent.

One of the best screening media currently available is that sold underthe trade name “Brilliance” MRSA Agar, available from Oxoid Limited.This is an opaque medium incorporating a chomogen that yields a bluecolour as a result of phosphatase activity. The enzyme phosphatase ispresent in many staphylococci, including S. aureus. Accordingly, toallow the medium to differentiate MRSA from other staphylococci, themedium also comprises a combination of antibacterial compounds selectedto inhibit the growth of a wide variety of likely competitor organisims,including meticillin-sensitive Staph. aureus (MSSA). Also incorporatedin the medium are compounds to suppress the expression of phosphataseactivity in other staphylococci, thus providing a high level ofsensitivity and specificity. MRSA, if present, are discernible by theirdistinctive bright blue colouration on the medium.

Although the current method and medium are satisfactory, it is stillnecessary to transfer the sample from the collection swab to a growthmedium and to incubate the plates at 37° C. for at least 18 hours afterinoculation with the swabs. The transfer step consumes the time of alaboratory technician, especially if screening is to be implemented on alarge scale, and increases the risk of contamination or other mishap.

Rambach et al (US2006/0035309) discloses a method for detectingmeticillin-resistant micro-organisms using a solid medium comprising acephalosporin and a chromogenic agent which releases a chromophore afterhydrolysis by an enzyme which is produced by the micro-organisms to bedetected. Rambach et al do not disclose the use of liquid or semi-solidmedia and do not disclose the use of an appropriate medium at thepoint-of-care.

Given the very large numbers of individuals whom it is now envisagedwill need to be screened, the average hospital laboratory will need touse many thousands of plates a year, and at any one time there arelikely to be several hundred plates requiring incubation or checkingwhich will take up a lot of volume in incubators or on desks.

Accordingly, there is an urgent need for a method of screening for MRSAwhich eliminates the labour-intensive transfer of samples fromcollection swab to growth medium (along with the risks associated withsuch transfer), is cost-effective even if performed on a large scale anduses less space.

SUMMARY OF THE INVENTION

The invention also thus provides a method of screening for the presenceof MRSA in a subject, the method comprising the steps of:

-   (a) obtaining a suitable sample from the subject using a swab or    other suitable sample collection device;-   (b) substantially immediately after performing step (a), contacting    the swab or other sample collection device with an identification    medium, said medium comprising:    -   (i) nutrients to allow the growth of MRSA;    -   (ii) at least one inhibitor substance to inhibit preferentially        the growth of organisms other than MRSA; and    -   (iii) a visual indicator substance which generates a visual        indication of the growth of MRSA if present;-   (c) incubating the identification medium, typically whilst still in    contact with the swab or other sample collection device, in    conditions which are sufficient to cause the growth of MRSA; and,    after sufficient time has elapsed;-   (d) inspecting the medium to determine the presence or absence of    the visual indication of the growth of MRSA; wherein both steps (a)    and (b) take place at the point of care (e.g. typically in the    presence of the subject).

The step (b) takes place substantially immediately after step (a). Thatis, in practice, it is desirable that both steps (a) and (b) take placeat the point of care, with the health worker swabbing the subject andthen substantially immediately contacting the swab or other samplecollection device with the identification medium. “Substantiallyimmediately” in this context means that after obtaining a sample fromthe subject, the swab or other sample collecting device is contactedwith the identification medium typically within 3 minutes, preferablywithin 2 minutes and most preferably within 1 minute.

The invention also thus provides a method of screening for the presenceof MRSA in a subject, the method comprising the steps of:

-   (a) obtaining a suitable sample from the subject using a swab or    other suitable sample collection device;-   (b) contacting the swab or other sample collection device with an    identification medium, said medium comprising:    -   (i) nutrients to allow the growth of MRSA;    -   (ii) at least one inhibitor substance to inhibit preferentially        the growth of organisms other than MRSA; and    -   (iii) a visual indicator substance which generates a visual        indication of the growth of MRSA if present;-   (c) incubating the identification medium, typically whilst still in    contact with the swab or other sample collection device, in    conditions which are sufficient to cause the growth of MRSA; and,    after sufficient time has elapsed;-   (d) inspecting the medium to determine the presence or absence of    the visual indication of the growth of MRSA; wherein both steps (a)    and (b) take place at the point of care (e.g. typically in the    presence of the subject).

The swab sample/medium combination will then typically be passed to amicrobiology lab, where incubation step (c) and inspection step (d) willnormally take place.

It is preferred that, once the swab has been contacted with the medium(step b), the swab is maintained in contact with the medium during atleast part, and preferably all, of step (c), although if desired forsome reason, the swab or other sample collection device can be removedfrom contact with the medium after a suitable time has elapsed. Further,once step (b) has occurred, it is preferred that the medium ismaintained at a temperature in the range 16-40° C., preferably 16-38°C., as soon as is practicable after the swab is contacted with themedium, and that step (c) should follow as soon as is practicable afterperforming step (b).

In a second aspect, the invention provides a kit for use in the methodsof the first aspect, the kit comprising: a sterile package containing aswab or other sample collection device, and an identification medium asdefined in the first aspect above.

The swab of use in the various aspects of the invention may be ofsubstantially conventional design, and is readily available commerciallyfrom many sources. The swab may be, for example, tipped with cotton,rayon, nylon, Dacron™, or flocked.

Advantageously, the swab is provided as an integral, but removable,component of a container used to contain the identification medium. Inuse, the swab component is removed from the container, contacted withthe relevant portion of the subject to be swabbed, and then reintroducedinto the container in such a way that the swab contacts the mediumtherein. The container will typically comprise a tube, with a volume inthe range 3-30 mls, preferably 5-25 mls, more preferably 10-25 mls,which can be accommodated in conventional laboratory racking for testtubes.

The identification medium of use in the invention will comprisenutrients sufficient for the growth of S. aureus, such as peptones,yeast extract, various salts and minerals, and a source of carbohydrate,such as glucose, dextrin or starch. The medium is preferably liquid, orsemi-solid, but could be solidified. For present purposes, a semi-solidmedium is one which is quasi-solid (i.e. can support its own weight andhold its shape, but has the ability to flow under pressure and willconform to the shape of a surface which applies pressure to it). Aquasi-solid medium can be obtained, for example, by utilising aconcentration of gelling agent (e.g. agar, agarose, Sephadex™ orgelatine) lower than that conventionally used to prepare solid media. Asan illustration, a concentration of agar in the range 1-8 grams perlitre, preferably 2-7 grams per litre, most preferably 3-6 grams perlitre, may be used to provide a quasi-solid medium.

Liquid and quasi-solid media are preferred since, inter alia, they allowfor greater contact of the medium with the swab than does a solidmedium.

The medium will further comprise at least one inhibitor substance toinhibit the growth of competitor organisms (i.e. those organisms otherthan MRSA). In practice, the medium is likely to comprise a plurality ofinhibitor substances. Such a plurality of inhibitor substances willnormally be required because of the variety of competitor organisms thatmay be present in the sample, which may include, for example, coagulasenegative staphylococci (CoNS), Gram negative organisms, Enterococcusspp., and yeasts. Accordingly the medium will typically comprise one ormore, preferably two or more, and most preferably three or more of thefollowing inhibitors: polymyxin B, colistin sulphate, aztreonam, lithiumchloride, deferoxamine mesylate, and amphotericin B, Fluconazole oranother antifungal compound.

In addition, because of the need to inhibit, in particular, the growthof MSSA, the medium will preferably comprise one or more of thefollowing; meticillin; oxacillin, a cephalosporin or a cephamycin.

The inhibitor substances are selected, and are present at aconcentration, such that they preferentially inhibit competitororganisms. That is, they inhibit the growth of competitor organisms to agreater extent than they inhibit the growth of MRSA.

Further, the identification medium will comprise at least one visualindicator substance, which generates, directly or indirectly, a visualsignal indicative of the growth of MRSA. Typically, but not necessarily,the visual signal will take the form of a colour change. This could be,for example, a change from one colour to a different colour; a changefrom colourless to a colour, or vice versa. In one embodiment, theindicator substance may comprise an indicator of pH change or redoxchange, such changes occurring as a result of the fermentation oralteration of a substrate within the medium, due to the action of one ormore enzymes elaborated by MRSA. Examples of such indicators include,for example, phenol red, neutral red or aniline blue.

In a preferred embodiment however, the indicator substance comprises achromogenic or fluorogenic compound. Preferably the indicator substanceis altered by the action of a phosphatase and/or a ribosidase producedby MRSA. Preferred chromogens are altered by the action of MRSAphosphatase to produce a coloured product. Particularly preferredchromogens for inclusion in the medium include indoxyl phosphates.Preferred fluorogens include umbelliferyl phosphate fluorogens, such as4-methylumbelliferyl phosphate.

In some embodiments, the medium may comprise a pH indicator as the“visual indicator substance”, or may comprise a fluorogen “visualindicator substance”, but not both a pH indicator and a fluorogen,because use of two indicators does not significantly increase the easeor sensitivity of detection, but rather adds to the cost and complexityof making the medium.

Finally, the medium may comprise a gelling agent such as agar,Sephadex™, agarose or gelatine, to provide a semi-solid medium, thusensuring the swab or other sample collection device remains in contactwith the medium.

The person skilled in the art will, with the benefit of the presentdisclosure, be able to determine appropriate concentrations for thevarious ingredients of the medium, and sample formulations are given inthe examples below.

The method of the invention involves obtaining a suitable sample fromthe subject. This will generally entail swabbing the nostrils and/or thehands of the subject, which are those sites most commonly colonised byMRSA, although other portions of the subjects's skin may also oralternatively be swabbed.

The method is conveniently performed using a kit in accordance with thesecond aspect of the invention. In one embodiment, the kit is providedas a sterile package, with an impermeable wrapping such as a metalisedfoil or a synthetic plastics film or bag, within which is provided aglass or synthetic plastics tube, bottle or other container housing analiquot of the growth/identification medium, sealed with a removablesealing means, such as a screw cap or other device. The kit will alsocomprise a label to accommodate relevant information, such as thesubject's name, the date of sampling, the site on the subject sampled,and so on. The label may be provided separately or may be providedalready attached to the tube, bottle or container housing the medium.Desirably a self adhesive label is used.

The package will also comprise one or more swabs such as a wooden orplastics—handled swab, or other suitable sample collection device. Thesewill be sterile (e.g. uv-irradiated) and may be packaged separately fromthe container housing the medium. In an alternative embodiment, a swabis provided as an integral part of the container. As an example, thecontainer may take the form of a tube with a screw cap. Projectingupwards from the cap is an integrally formed swab. To obtain a sample,the cap is unscrewed from the tube, and the swab contacted with thesubject. The cap is then replaced in the tube in the reverseorientation, with the swab projecting downwards into the medium. Ifdesired the cap can be provided with a screw-threaded portion in anopposite sense, to assist in replacing the cap on the tube in thereverse orientation. Alternatively, when inserted into the container inthe reverse orientation the cap may form a seal by means of a stopper orbung effect.

Once contacted with the medium, the swab could in principle be removedafter agitation. However, it is preferable to retain the swab in contactwith the medium during the incubation step to ensure that at least someof the MRSA bacteria present are transferred to the medium.

The incubation conditions will be apparent to those skilled in the artand will usually involve incubation at a temperature above ambient,preferably in the range 30-40° C., and most preferably at about 37° C.The length of time required in order to produce a visual signalindicative of the presence of MRSA will vary depending on the preciseconditions of the assay, the composition of the medium, and thephysiological state of the organisms at the time of sampling (e.g.whether or not physiologically stressed and to what extent). Typicallyan incubation of about 18-24 hours is appropriate, but in someconditions the inventors believe it may be possible to obtain apresumptive result after as little as 6 hours' incubation in step (c).

The medium will most conveniently be directly inspected by a humanobserver, but if desired an automated system could be employed, themedium being interrogated by a machine such as a spectrophotometer orother colour or fluorescence-sensitive device.

The method of the invention offers four principle advantages overconventional prior art methods.

Firstly, in the prior art, a swab sample is taken from a patient andthen transferred to the hospital lab either in a sterile containerwithout medium or in a sterile transfer medium (such as saline) whichdoes not promote the growth of MRSA. Once the swab reaches thelaboratory, no immediate action may be taken: the swab may be held on abench and batched until a sufficient number have been received in orderto justify a technician's time in using the swabs to inoculate plates.

In contrast, with the method of the invention, the swab is immediatelyplaced in contact with a medium which promotes and stimulates the growthof MRSA, whilst simultaneously preferentially inhibiting the growth ofcompetitor organisms. This gives the MRSA organisms a “head start”relative to the prior art method, such that results can be obtained morerapidly after swabbing of the subject has taken place.

Secondly, no manipulation of the samples is required when received inthe laboratory, and they are therefore less likely to be delayed beforeprocessing. Instead, a technician simply has to place the sample tube orcontainer in an incubator. Reducing the number of manipulations of thesamples also reduces the risk of contamination.

Thirdly, the tubes or containers employed in the present inventionoccupy far less volume than conventional plates, thus saving on desktopspace and incubator space, which is very significant, when large numbersof samples are involved. Petri dishes, used for making plates of solidculture media, are usually 90-150 mm in diameter, whereas the tubes orcontainers for use in the present method are typically only 10-20 mm indiameter.

Fourthly, the method of the present invention should provide greatersensitivity than conventional methods. Where a swab is taken and sent tothe laboratory in isolation (i.e. not in contact with a liquid orsemi-solid medium), the organisms present on the swab will be insub-optimal conditions. Some may die, and even if the organisms do notdie they will become physiologically stressed, which may mean they areoutgrown by more robust competitor organisms when the swab is used toinoculate a solid growth medium, leading to a false negative result.

Even if the swab is inserted into a conventional transport medium (suchas Amies medium), the conditions will still be sub-optimal. By way ofexplanation, transport media are intended to hold microorganisms in astate of relative ‘stasis’, such that the number of viable cellssubsequently recovered reflects that contained within the specimen attime of collection. This means that any delay between sampling andinoculation of a growth medium does not cause an increase in numbers ofthe target organisms. Sensitivity of the final growth medium isdependent on the number of viable cells remaining in the transport swabat the point of inoculation.

In contrast, the method of the invention ensures that the number oforganisms in the specimen start to increase as soon as it is placed intothe differential MRSA transport medium, provided that the medium remainsat a temperature between 16-40° C., preferably 16-38° C. As the targetorganisms are provided with the appropriate nutrients, the number ofviable cells is likely to increase. Similarly, non-target organisms willbe inhibited as soon as the specimen is placed into the MRSA transportmedium, reducing the likelihood of over-growth of ‘background flora’ andresulting competitive inhibition of MRSA. This increases sensitivity ofthe method.

Finally, the method of the invention is labour saving. The currentpractices in microbiology laboratories involve inoculation of ascreening swab (usually nasal swab taken from a patient and contained ina transport medium) onto a solid or liquid growth medium. The growthmedium is then incubated to stimulate growth of MRSA. This practice iscarried out by laboratory staff. Many swabs are received by laboratorieseach day for MRSA screening, making this a labour-and time-intensiveprocedure.

In a third aspect the invention provides an identification mediumsuitable for the screening of samples from a subject for the presence ofMRSA, the medium being liquid or semi-solid and comprising:

-   -   (i) nutrients to allow the growth of MRSA;    -   (ii) at least one inhibitor substance to inhibit preferentially        the growth of organisms other than MRSA; and    -   (iii) a visual indicator substance which generates a visual        indication of the growth of MRSA if present.

The medium is preferably initially colourless and translucent, andbecomes coloured if MRSA organisms grow during incubation of the medium.Other preferred features of the medium are as recited above in thecontext of the first and second aspects of the invention.

The invention will now be further described by way of illustrativeexample, and with reference to the accompanying drawing FIG. 1, which isa flow chart comparing the prior art method with the method of theinvention.

EXAMPLE 1 A Liquid (Broth) Medium Formulation Suitable for Use in theVarious Aspects of the Invention

The liquid medium will contain the following:

a nutritive broth base, such as tryptone soy broth, nutrient broth;

at least one substance to inhibit the growth of organisms other thanMRSA, such as meticillin, oxacillin, various cephalosporins orcephamycins;

at least one substance to inhibit the growth of organisms other thanStaphylococcus aureus. Such substances include polymixin B, colistinsulphate, aztreonam, lithium chloride, deferoxamine mesylate,amphotericin B, fluconazol, anisomycin or other antifungal compounds;and

at least one indicator compound to differentiate the growth of MRSA fromother organisms. This can be an indicator of pH or redox changeresulting from fermentation of carbohydrate contained within saidmedium. Such indicators could include phenol red, neutral red,bromocresol purple, bromothymol blue, or aniline blue.

In another example, the indicator can be substituted for at least onechromogenic compound to detect enzyme activity specific toStaphylococcus aureus. Examples of this are indoxyl phosphate chromogenswhich detect phosphatase activity.

In another formulation, the indicator can be substituted for at leastone fluorogenic compound to detect enzyme activity specific toStaphylococcus aureus. An example of this is the 4-methylumbelliferylphosphate fluorogen which detects phosphatase activity.

One sample formulation is as follows:

Typical composition per litre of medium:

Peptone 10-15 g Beef Extract 1-5 g Mannitol 5-15 g Sodium chloride 50-75g Phenol Red 20-30 mg Oxacillin 4-6 mg Polymixin B 10-15 mg Aztreonam10-25 mg

EXAMPLE 2 A Semi-Solid Medium Formulation Suitable for Use in theVarious Aspects of the Invention

The semi-solid medium will contain the following:

a nutritive broth base, such as tryptone soy broth or nutrient broth,containing at least one gelling agent. Such gelling agents could includeagar, agarose, Sephadex™ or gelatine. A concentration range would berequired to provide a gel strength which prevents spillage of the mediumfrom the culture tube or bottle, and enables optimal contact of themedium with the swab. One example is a concentration of agar between 1and 8 grams per litre;

at least one substance to inhibit the growth of organisms other thanMRSA, such as meticillin, oxacillin, various cephalosporins orcephamycins;

at least one substance to inhibit the growth of organisms other thanStaphylococcus aureus. Such substances include polymixin B, colistinsulphate, aztreonam, lithium chloride, deferoxamine mesylate,amphotericin B, fluconazole, anisomycin or other antifungal compounds;and

at least one indicator compound to differentiate the growth of MRSA fromother organisms. This can be an indicator of pH or redox changeresulting from fermentation of carbohydrate contained within saidmedium. Such indicators could include phenol red, neutral red,bromocresol purple, bromothymol blue, or aniline blue.

One sample formulation is as follows:

Typical composition per litre of medium:

Peptone 10-15 g Beef Extract 1-5 g Mannitol 5-15 g Sodium chloride 50-75g Agar 2-8 g Phenol Red 20-30 mg Oxacillin 4-6 mg Polymixin B 10-15 mgAztreonam 10-25 mg

In another example, the indicator can be substituted for at least onechromogenic compound to detect enzyme activity specific toStaphylococcus aureus. Examples of this are indoxyl phosphate chromogenswhich detect phosphatase activity.

In another example, the indicator can be substituted for at least onefluorogenic compound to detect enzyme activity specific toStaphylococcus aureus. An example of this is the 4-methylumbelliferylphosphate fluorogen which detects phosphatase activity.

EXAMPLE 3

As shown in the Flow diagrams in FIG. 1, the method of the invention(timeline on the right hand side) provides a more streamlined processthan the prior art method (timeline on the left hand side) and, inpractice, gives rise to a presumptive result several hours in advance ofthe conventional prior art technique.

1. A method of screening for the presence of MRSA in a subject, themethod comprising the steps of: (a) obtaining a sample from the subjectusing a swab or other sample collection device; (b) contacting anidentification medium with the swab or other sample collection devicecontaining the sample, said medium comprising: (i) nutrients sufficientto allow the growth of MRSA if present; (ii) at least one inhibitorsubstance to inhibit preferentially the growth of organisms other thanMRSA; and (iii) a visual indicator substance which generates a visualindication of the growth of MRSA if present; (c) incubating theidentification medium, after or while still in contact with the swab orother sample collection device, in conditions sufficient to cause thegrowth of MRSA if present; and (d) inspecting the medium to determinethe presence or absence of the visual indication of the growth of MRSA.2-21. (canceled)
 22. The method according to claim 1, wherein theidentification medium is liquid or a semi-so lid.
 23. The methodaccording to claim 1, wherein steps (a) and (b) are performed at thepoint of care of the subject.
 24. The method according to claim 1,wherein the medium comprises a source of carbohydrate utilizable as anenergy source by MRSA and one or more of the following sources ofnutrients: peptones; yeast extract; salts; and minerals.
 25. The methodaccording to claim 1, wherein the medium comprises two or more of thefollowing substances at a concentration sufficient preferentially toinhibit the growth of competitor organisms: polymyxin B; colistinsulphate; aztreonam; lithium chloride; deferoxamine mesylate; andamphotericin B, fluconazole or another antifungal compound.
 26. Themethod according to claim 25, wherein the medium comprises three or moreof the inhibitor substances recited in claim 5, at a concentrationsufficient preferentially to inhibit the growth of competitor organisms.27. The method according to claim 1, wherein the medium comprises, at aconcentration sufficient preferentially to inhibit the growth of MSSA orother competitor organisms if present, one or more of the following:meticillin; oxacillin; a cephalosporin; and a cephamycin.
 28. The methodaccording to claim 27, wherein the medium comprises, at a concentrationsufficient preferentially to inhibit the growth of MSSA or othercompetitor organisms, two or more of the antibacterial substancesrecited in claim
 7. 29. The method according to claim 1, wherein theindicator substance comprises a chromogen or a fluorogen.
 30. The methodaccording to claim 1, wherein the indicator substance comprises achromogen or fluorogen substrate for MRSA phosphatase.
 31. The methodaccording to claim 1, wherein step (c) comprises incubating the medium,after or while still in contact with the swab or other sample collectiondevice, at a temperature in the range of 35-38° C. for a time period inthe range of 16-24 hours.
 32. The method according to claim 1, whereinthe visual indicator substance generates a signal specificallyindicating the growth of MRSA.
 33. The method according to claim 1,comprising use of a medium which comprises a pH indicator or afluorogen, but not both.
 34. A kit for use in performing the method ofclaim 1, the kit comprising a sterile package containing a swab or othersample collection device and an identification medium as recited inclaim
 1. 35. The kit according to claim 34, wherein the identificationmedium is provided in liquid or semi-solid form and is contained withina sterile container having a removable and resealable sealing means. 36.The kit according to claim 35, wherein the container comprises a screwcap.
 37. The kit according to claim 35, wherein the swab or other samplecollection device is provided as an integral part of the container. 38.The kit according to claim 34, further comprising instructions forperforming the method of claim
 1. 39. The kit according to claim 34,comprising a medium which comprises a pH indicator or a fluorogen, butnot both.
 40. A liquid or semi-solid identification medium suitable foruse in performing the method of claim 1, the medium being as recited inclaim
 1. 41. The medium according to claim 40, the medium comprising apH indicator or a fluorogen, but not both.